Comparing CB1 receptor GIRK channel responses to receptor internalization using a kinetic imaging assay

The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the β-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The β-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid’s GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.


GIRK channel assay and CB1R internalization assay
GIRK channel activation was monitored in the 96-well clear-bottom plates by recording cell membrane potential (MP) via fluorimetry as previously described 25,26 .For the MP measurements, the AtT20-SEPCB1 cells were incubated for 30 min in a buffer solution consisting of 132 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 ,5 mM dextrose, 5 mM HEPES, pH 7.4 (with NaOH), with MPSD (FLIPR Membrane Potential kit RED; MolecularDevices).Prior to the fluorescence measurements, the cells were loaded with MPSD in buffer solution (132 mM NaCl, 1 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 ,5 mM dextrose, 5 mM HEPES, pH 7.4 (with NaOH)as above) and incubated for an additional 5 min.Fluorescent signals were recorded using a SynergyHT Cytation microplate reader (Biotek) at 28 °C25,26 .(±) CP55,940 and WIN55,212-2 were dissolved in DMSO at stock concentrations of 100 mM, AEA was dissolved in ethanol (as prepared by Cayman chemical), and Δ 9 -THC was dissolved in acetonitrile (as prepared by Cayman Chemical).The stock concentration was serially diluted for all cannabinoids in 1 mM KCl buffer solution containing the MPSD to create the working concentrations.The cannabinoids or control solution (20 μL) were injected into each well (total volume = 220 μL) at time zero.Data were collected at 9 s intervals from 36 s before compound addition until 240 s after compound addition (Fig. 1) at excitation and emission wavelengths of 520 and 560 nm, respectively.

CB1R imaging
CB1R internalization was recorded in 96-well plates by imaging AtT20-SEPCB1R expression on the cell surface.pHluorin is a pH-sensitive green fluorescent protein whose cell surface fluorescence can be visualized at 525 nm.Because FBS increases background fluorescence and decreases image clarity, the FluoroBrite media used for cell culture was replaced with 100 μL FluoroBrite media containing 1% Pen-Strep, 2% Glutamax, 10 mM HEPES, and no FBS (Imaging media).Stock solutions of cannabinoids were diluted in imaging media to working concentrations.CB1R inverse agonist/antagonist, SR141716 was diluted in DMSO to a stock concentration of 3 mM, then diluted in imaging media.Images of AtT20-SEPCB1 cells were taken at 40 × using a BioTek Cytation 5 microplate reader (Agilent) at 28 °C with excitation and emission wavelengths 469 and 525 nm, respectively.

CB1R image and animation generation
Visualization of CB1R internalization was conducted using ImageJ/FIJI software version 2.15.1, as above.Z-stacks were compressed into 1 image per time point set to maximal fluorescence.Each set of images were then z-stacked and aligned across all time points (see representative videos in Supplementary files).Images were background subtracted, and then a FIRE look-up table (LUT) was applied to represent change in fluorescent intensity.These images were not used for data analysis.

Statistical analysis
Data from GIRK channel assays were fit to a one-site exponential decay curve in GraphPad Prism (version 9.0) to estimate the rate of GIRK channel response (Supplementary Fig. 1).Data from GIRK channel assays were also analyzed using the Area Under the Curve function with default settings in GraphPad Prism.Peak F/F 0 readings at 240 s for each cannabinoid were plotted against compound concentration (Supplementary Fig. 2).AUC and peak F/F 0 data were then normalized to the (±)CP55,940 maximum and fit to the four-parameter, non-linear regression analysis in Graphpad Prism (v.9.0): y = y min + (y max − y min /1 + 10^ ((LogEC 50 -Log Concentration), where EC 50 is the concentration producing a 50% increase in the maximal response y max (E max ), and y min is defined as a minimum fluorescent response.The same data analysis procedure was followed for CB1R internalization data using the one-site exponential decay curve for rate of internalization (Supplementary Fig. 3), AUC analysis (Supplementary Fig. 4) and subsequent concentration-response curve analyses.Data are presented as the mean ± standard error of the mean (S.E.M.).Statistical analyses were one-or two-way analysis of variance (ANOVA) followed by Tukey's or Dunnett's post-hoc tests (one-way ANOVA) or Bonferroni's post-hoc test (two-way ANOVA), respectively and as indicated.P < 0.05 was considered statistically significant.Compound treatment replicates or individual cells are represented by n values, as indicated in figure legends.

CB1R internalization can be imaged and quantified using AtT20-SEPCB1 cells
Establishing the CB1R internalization assay The CB1R internalization experiments followed the GIRK1/2 channel assay protocol modified for imaging.AtT20-SEPCB1 cells were cultured in clear-bottom, black-walled, 96-well plates, with two wells being vehicle controls and the rest treated with the compounds.Cells were recorded at 40 × magnification at 5 min intervals following treatment with vehicle, (±)CP55,940, WIN55,212-2, Δ 9 -THC, or AEA (see Supplementary video files for Fig. 3), and a false-color heat map was applied to images to visualize SEP-CB1R in video montages (Fig. 3a-e).Most AtT20 cells expressed GFP labeling, thus confirming stable transfection with the SEPCB1 construct (Fig. 3a [lower panel]).

Synthetic cannabinoids potently induce CB1R internalization
CB1R internalization was imaged and measured for (±)CP55,940, WIN55,212-2, Δ 9 -THC, and AEA (Fig. 4a-d).WIN55,212-2 produced the greatest CB1R internalization, whereas Δ 9 -THC produced the least (Fig. 4c,e).As was done for the GIRK channel assay, peak CB1R internalization at 30 min was plotted against concentration, and rank order potency was determined to be WIN55,212-2 > (±)CP55, 940 > Δ 9 -THC > AEA (Fig. 4f, Table 1).The rank order efficacy was (±)CP55, 940 > WIN55,212-2 > AEA > Δ 9 -THC (Fig. 4f, Table ).These rank orders of potency and efficacy were the same as observations made in the GIRK channel assay.However, no statistically significant differences were detected between (±)CP55, 940 and other compounds in the CB1R internalization assay.In general, cannabinoid potency was less in the CB1R internalization assay than in the GIRK assay, although these differences were not statistically significant (as determined by two-way ANOVAs followed by Bonferroni's post-hoc test).Co-treatment of cells with 1 µM WIN55,212-2 and 1 µM SR141716 reduced CB1R internalization, indicating the quantification approach used was measuring CB1R trafficking (see Supplementary video files for Fig. 5; Fig. 5).SR141716 was not assessed alone in these experiments and therefore the reason that this antagonist did not fully reverse WIN55,212-2 mediated CB1R internalization is not clear.Further assessment of CB1R trafficking in response to antagonists and inverse agonists with this model system is needed.As with the GIRK channel response, the kinetics of CB1R internalization may depend on the cannabinoid bound to CB1R.No significant change in the slope was observed when responses were compared within each compound tested (Supplementary Fig. 3a-d).When the slope was compared between these maximum responses, the rate of CB1R internalization was not significantly different between cannabinoids (Supplementary Fig. 3e).The AUC was calculated for each CB1R internalization response and graphed against each cannabinoid concentration (Supplementary Fig. 4).These data were fit to a four-parameter non-linear regression.In this analysis, the rank order potency and efficacy were not different from our calculations using peak CB1R internalization response at 30 min (Supplementary Fig. 4).

Discussion
Studies into biased signaling and receptor-ligand binding highlight the diversity of cannabinoid-CB1R molecular signaling [27][28][29] .This research targets the GIRK1/2 channel and CB1R internalization responses of four cannabinoids: (±)CP55,940, WIN55,212-2, AEA, and Δ 9 -THC.In this study, the synthetic cannabinoids (±)CP55,950 and WIN55,212-2 were more potent and efficacious at stimulating a GIRK1/2 channel response than AEA and Δ 9 -THC, aligning with previous research 25,30 .Specifically, the trace of (±)CP55,940's GIRK1/2 response significantly differed from the other cannabinoids, suggesting different GIRK1/2 channel kinetics (Fig. 2e and Supplementary Fig. 1e).Of note, GIRK channel responses to these cannabinoids were not tested in cells lacking CB1R; therefore, Table 1.GIRK channel responses and CB1R internalization in AtT20 cells following cannabinoid treatment.AtT20 cells stably-expressing SEP-CB1R were treated with 10 fM -10 μM of cannabinoids as indicated and GIRK channel response was measured continuously for 6 min (i.e., 240 s) with the mean time courses shown in figure 2a-e, or CB1R internalization was measured at 5 min intervals for 30 min with the mean time courses shown in figure 4a-e.Here, peak responses at 240 s (GIRK) and 30 min (internalization) for each compound were plotted against log[Compound], (M) and normalized to the maximal (±)CP55,940 response (i.e., 100%).Data were fit to a four-parameter non-linear regression (Fig. 2f, 4f) to estimate potency and efficacy.All data are presented as mean ± S.E.M. *p<0.05,**p<0.01,****p<0.0001compared to (±)CP55,940 as determined by one-way ANOVA within assay followed by Dunnett's post-hoc test.non-cannabinoid receptor effects on GIRK channels by these ligands can not be ruled out in our findings.Synthetic cannabinoids have been shown to form stronger interactions within the CB1R binding pocket, which may induce conformational changes that promote G i signaling 28,31,32 .Importantly, this study focused on AEA and did not include 2-arachidonoylglycerol, which has been described elsewhere as more potent and efficacious than AEA 23 ; future studies should compare these two endocannabinoids for differential responses in these assays.Phosphorylation of the CB1R by specific G protein-coupled receptor kinases (GRKs) aids in the recruitment of β-arr2, which then blocks the reassembly of the Gαβγ i complex, leading to receptor desensitization and internalization 23,33,34 .Supporting the link between β-arrestins 1 and 2 and CB1R internalization is research by Flores-Otero et al., who found WIN55,212-2 recruits β-arr in parallel with CB1R internalization 15 .Research has also demonstrated that the CB1R internalization response varies depending on the cannabinoid 35 .In this study, the synthetic cannabinoids ranked higher in potency than AEA and Δ 9 -THC.In line with this, Δ 9 -THC binds to the CB1R in such a way that it forms a less stable active confirmation than synthetic cannabinoid receptor agonists 28,36 .Interestingly, AEA was more effective at inducing CB1R internalization compared to (±)CP55, 940, albeit this difference was not statistically significant and with lower potency.Similar to Δ 9 -THC, AEA is proposed to have unstable interactions with residues promoting CB1R-G i signaling as opposed to CP55,940, which produce confirmation changes favorable to G i signaling 28,29 .Sites implicated for β-arr2 recruitment and CB1R internalization include the c-terminus and transmembrane helices 2 (TMH2) and 7 (TMH7), whereas site such as α5 and intracellular loop 2 (ICL2) are important for CB1R-G i 23,28,37 .While different sites on the CB1R proposed for β-arr2 and Gα i functions exist, research has shown that GIRK1/2 channel function and CB1R internalization are mediated by the same amino acid residues on the CB1R 38 .In AtT20 cells, a D164N mutation on TM2 inhibited CB1R internalization and potentiation of GIRK channel current 34,38 .When comparing a cannabinoid's GIRK1/2 channel assays and CB1R internalization results, no significant differences were found between potency and efficacy.These results suggest that when a cannabinoid binds to the CB1R, the effects of the Gβγ i signaling and β-arr2 recruitment are balanced.It is worth noting that within the GIRK1/2 channel assay, there were significant differences between the GIRK1/2 channel potency and efficacy of (±)CP55,940 compared to AEA and Δ 9 -THC; however, when repeated with CB1R internalization, no significant differences were observed.We considered that the significant differences found in the GIRK1/2 channel assay did not translate to the CB1R internalization assay because we measured peak responses at different time points.The first wave of CB1R intercellular signaling occurs rapidly and is primarily G i -driven, whereas peak β-arr2 occurs approximately 20 min later 14,16 .Peak GIRK1/2 channel and CB1R internalization responses were determined within the appropriate time frames; therefore, the lack of significant differences in the CB1R internalization assay is unlikely due to its peak response being missed at an earlier time point.This may be due to variability in the internalization assay, such that the error was too large to detect a statistically significant

Figure 1 .
Figure 1.Experimental timeline for CB1R GIRK channel response quantification and internalization imaging.GIRK channel and CB1R internalization experiments were run separately but compared here for reference.Baseline images of AtT20-SEPCB1 cells were taken 5 min before exposure to a cannabinoid.Basal GIRK channel activity was recorded for 36 s prior to compound injection.The change in fluorescent signal, representing CB1R surface expression, was imaged at compound injection and every 5 min thereafter for 30 min.GIRK channel responses were recorded every 9 s for 240 s (i.e., 6 min) after compound exposure.